Affected by Different Forms of Foliar Zinc Fertilization
Yanyan Wei., M. J. I. Shohag., Xiaoe Yang*
Ministry of Education (MOE) Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang University, Hangzhou, People’s Republic of China
Abstract
Background:Zinc (Zn) biofortification through foliar Zn application is an attractive strategy to reduce human Zn deficiency. However, little is known about the biofortification efficiency and bioavailability of rice grain from different forms of foliar Zn fertilizers.
Methodology/Principal Findings:Four different Zn forms were applied as a foliar treatment among three rice cultivars under field trial. Zinc bioavailability was assessed byin vitrodigestion/Caco-2 cell model. Foliar Zn fertilization was an
effective agronomic practice to promote grain Zn concentration and Zn bioavailability among three rice cultivars, especially, in case of Zn-amino acid and ZnSO4. On average, Zn-amino acid and ZnSO4increased Zn concentration in polished rice up
to 24.04% and 22.47%, respectively. On average, Zn-amino acid and ZnSO4increased Zn bioavailability in polished rice up to
68.37% and 64.43%, respectively. The effectiveness of foliar applied Zn-amino acid and ZnSO4were higher than Zn-EDTA
and Zn-Citrate on improvement of Zn concentration, and reduction of phytic acid, as a results higher accumulation of bioavailable Zn in polished rice. Moreover, foliar Zn application could maintain grain yield, the protein and minerals (Fe and Ca) quality of the polished rice.
Conclusions:Foliar application of Zn in rice offers a practical and useful approach to improve bioavailable Zn in polished rice. According to current study, Zn-amino acid and ZnSO4 are recommended as excellent foliar Zn forms to ongoing
agronomic biofortification.
Citation:Wei Y, Shohag MJI, Yang X (2012) Biofortification and Bioavailability of Rice Grain Zinc as Affected by Different Forms of Foliar Zinc Fertilization. PLoS ONE 7(9): e45428. doi:10.1371/journal.pone.0045428
Editor:Keqiang Wu, National Taiwan University, Taiwan
ReceivedJune 6, 2012;AcceptedAugust 17, 2012;PublishedSeptember 20, 2012
Copyright:ß2012 Wei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This research work was financially supported by the HarvestPlus-China Program (8234), International Cooperation Program from the Ministry of Science and Technology, China (2006AA06Z386), Project from the Zhejiang Provincial Department of Education, Zhejiang, China (20100339) and Fundamental Research Funds for the Central Universities (2012FZA6008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: xyang571@yahoo.com
.These authors contributed equally to this work.
Introduction
Zinc (Zn) deficiency is a well documented global health problem, affecting nearly half of the world population, particular in developing countries, where high proportion of cereal crops, such as rice and wheat, consumed as a staple food [1,2]. The reliance on cereal based food induce Zn deficiency related health problem, such as impairments in physical growth, immune system and brain function [3,4]. Among the cereals, Rice (Oryza staviaL.), being one of the leading staple crop for half of the world’s population and, hence, is the main source of Zn to human [5]. Rice, however unfortunately, is a poor source of metabolizable Zn, due to inherently low in Zn content and the bioavailable Zn [6]. Enrichment of rice with high bioavailable Zn is, therefore, suggested as a way to generate major health benefits for a large number of susceptible people.
Zinc biofortification, which aims to enhance Zn concentration as well as bioavailability of rice grain, is considered as the more sustainable and economical solution to address human Zn deficiency [7]. Genetic biofortification and agronomic
biofortifica-tion are two important agricultural tools to improve rice grain Zn concentration [2,8]. However, yield factor, interactions between genotype and environment, lack of sufficient genetic diversity in current cultivars for breeding program, consumer resistance and safety of genetically modified crops are the main bottlenecks of genetic biofortification [2,9,10,11]. The traditional and efficient strategy of agronomic biofortification, such as Zn fertilization is, therefore, urgent, essential and rapid solution for improving Zn concentration in rice grain to address the ongoing human Zn deficiency.
the rice grains through the phloem [16] and several members of the Zn-regulated transporters regulate this process [17]. In most of those literatures, the reported data are mostly based on brown rice. As polished rice is the main consumed portion by human, rare information was found on Zn concentration in polished rice after foliar Zn fertilizations. Moreover, time of foliar application and the different forms of foliar Zn fertilizers may differentially influence grain Zn concentration. In recent past, several studies have been conducted to adjust time of foliar Zn application in cereal crops [2,13,18]. It is now well established that foliar Zn application after flowering stage (e.g., at early milk plus dough stages) more distinctly increase the grain Zn concentration [13]. On the other hand, different Zn fertilizers such as inorganic and organic Zn salts play a fundamental role in the way in nutrient transport from leave to the grain [19]. Unfortunately, studies evaluating the effective-ness of foliar application of different Zn forms on rice grain Zn accumulation are still rare.
The metabolizable Zn from biofortified crop grain not only depends on net Zn concentration, but also a large extent on the bioavailability of Zn. Zinc bioavailability defined as the proportion of the total amount of Zn that is potentially absorbable in a metabolically active form [20]. Phytic acid, the naturally occurring anti-nutrient presents in the seed, reduces the bioavailability of Zn, because of its ability to form complex with Zn, and inhibits Zn solubility, digestibility and absorption in human body [21]. Although, it is assume that foliar Zn fertilization improved Zn bioavailability, but till now there are rare studies on the Zn bioavailability of rice grain deserved from different forms of foliar Zn application [2]. Hence, an in vivo approach to assess the potential benefits of different forms of foliar Zn application on grain Zn bioavailability is required.
Ideally, Zn bioavailability in crop grains should be evaluated through in vivo human study. However, complexity to perform large-scale screening of sample and cost limit their applicability [22].In vitrodigestion/Caco-2 cell model has been proposed as an alternative toin vivomethod for estimating mineral bioavailability in diets. In recent years, in vitro digestion/Caco-2 cell culture model is being utilized for absorption studies involving Zn. Thisin vitromodel is currently considered as the best approach, in term of cost and time, to investigate the bioavailability of different food components as a prelude toin vivo study [23]. The present study used this model to assess the bioavailability of Zn from polished rice grain fortified with different forms of foliar Zn fertilization.
Viewing the above circumstances, the current study were aimed: (i) to assess the effect of different forms of foliar Zn fertilizer on Zn concentration in brown rice and polished rice, (ii) to assess the effect of different forms of foliar Zn fertilizer on Zn bioavailability in polished rice. The findings of the current study were used to design experiment to identify some useful foliar Zn fertilizer for increasing the level of bioavailable Zn in rice grain.
Materials and Methods
Field Experiment and Sampling
Field location. Experimental site was Longyou, Zhejiang province (29u 029 N, 119u 119 E), China. The climate of the experimental site is subtropical humid. The soil type of exper-imental field was periodical water logged paddy soil. Before the start of experiments, puddle layer (0–15 cm top soil) soil samples were taken from four random spots of the field and analyzed for various physico-chemical properties (Table1).
Experimental design and treatment. Experimental design was a split plot with four replications. Foliar Zn fertilization treatments were treated as main plot and rice cultivars as sub-plot.
Foliar Zn fertilization treatments comprised of four different forms of Zn fertilizer: (i) ZnNa2EDTA (Zn-EDTA), Zn-EDTA was the
common Zn fertilizer which contain 9% Zn (ii) Zn-Citrate, in which Zn content was 10% (iii) ZnSO4?7H2O (ZnSO4), common
Zn fertilizer, in which Zn content was 36% (iv) Zn-amino acids (Zn-AA), Zn-AA contains Zn as ZnSO4 (10%) and amino acid
(25%) and, (v) Control, sprayed with distilled water. Three rice (Oryza sativa L.) varieties differ in their grain Zn concentration namely Hai7, Bing91185 and Biyuzaonuo were selected according to our previous study [24]. Thus, there were 60 plots with each 4 m2(262 m).
Thirty days old seedlings of each cultivar were transplanted to the plot. Before transplanting, the standard recommended dose of NPK fertilizer was applied to all plots at rates of 187.5 kg N ha21 (70% applied as basal dose and 30% as topdressing at panicle initiation stage), 70 kg P2O5 ha21and 93 kg K2O ha21. Water
management was the same as conventional rice farming practice. The foliar Zn was applied three times, one time at panicle initiation stage, two times at 7 days after flowering stage. Spray was applied after sunset. During spray, soil surface was covered to minimize the contamination of soil with foliar applied Zn. The concentration of Zn fertilizer was 0.2%. The amount of foliar Zn applied was equivalent to 2.5 kg Zn ha21. All foliar sprays contained 0.01% (v/v) Tween80 as a surfactant.
Rice sample preparation. Plants were harvested from the center of each plot at maturity and were manually threshed to separate grains. Rice grains were air dried; the brown rice was prepared by removing the husk using a laboratory de-husker (JLGJ4.5, Taizhou Cereal and Oil Instrument Co. Ltd., Zhejiang, China), the polished rice was prepared by polishing the bran by a laboratory polishing machine (JNMJ3, Taizhou Cereal and Oil Instrument Co. Ltd., Zhejiang, China). The rice samples were powdered to make flour by using a ball mill (Retsch, MM-301, Germany), then put in the plastic bag and keep at220uC until analysis. A part of rice was cooked for 15 min with 1:2 rice/ deionized water (w/v). The cooked rice samples were then homogenized in a polytron homogenizer and then the homoge-nates were frozen and lyophilized before testing via the in vitro
digestion/Caco-2 cell model.
Chemical Analysis
Mineral concentration determination. The ground rice samples (0.3 g) of each treatment were placed in to PTFE digestion tube and, digested with nitric acid (2 mL) and hydrogen peroxide (0.5 mL). After cooling, the digestion solution was transferred to a 25 mL volumetric flask, made up the volume
Table 1.Selected physical and chemical properties of the soils.
Characteristics Value
pH (H2O, 206C) 5.8
Total N (g kg21) 1.36
Organic matter (g kg21) 13.70
Olsen P (mg kg21) 42.6
CaCO3(%) 1.98
NH4OAC-exchangeable K (mg kg21) 90.35
DTPA-extractable Zn (mg kg21) 3.84
DTPA-extractable Fe (mg kg21) 198.45
with deionized water. The concentrations of Zn, iron (Fe), calcium (Ca) in sample were determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500a, Agilent Technologies, CA, USA) following our previously described method [16].
Phytic acid determination. Phytic acid from the rice samples was determined by the method described by Dai et al., [25]. Briefly, 0.5 g of rice flour was extracted with 10 mL of 0.2 M HCl for 2 h by a rotary shaker and then centrifuged at 10000gfor 10 min. The clear supernatant was collected, and 2 mL of 0.2% FeCl3was added to 2.5 mL of supernatant. The resulting solution
was mixed thoroughly, heated in a boiling water bath for 30 min, cooled in room temperature, and centrifuged at 10000g for 15 min. Then supernatant was discarded and the residue in the tube washed three times with 5 mL of deionized water. The tube was then centrifuged again at 10000g for 10 min after adding 3 mL of 1.5 M NaOH to it. The supernatant was discarded again, and 3 mL of 0.5 M HCl was added to the tube to dissolve the residue. Finally, deionized water was added to the solution made up to the volume of 10 mL. The Fe concentration in the solution was measured by ICP-MS (Agilent 7500a, Agilent Technologies, CA, USA). The phytic acid content was calculated by multiplying Fe content by the factor 4.2.
Protein content determination. Rice samples were ana-lyzed for protein content by determination of total nitrogen. The Kjeldahl method was used to determine total nitrogen. Rice samples (0.5 g) were digested by 20 mL H2SO4, and then distilled
in KjelFlex K-360 (Buchi, Flawil, Switzerland) with 40% (w/v) NaOH and 2% (w/v) boric acid (methyl red and bromcresol green used as an indicator solution), then titrated with 0.02 mM H2SO4.
The protein content was subsequently calculated by multiplying nitrogen content by a conversion factor of 5.95 [26].
Zinc Bioavailability Assay
In vitro digestion of rice sample. The in vitro digestion method according to our previously described method [27]. Briefly, 5 g of cooked rice powder was mixed with 15 mL of saline buffer (140 mM NaCl, 5 mM KCl), and pH was adjusted to 2 with 6 M HCl. Then, the sample was mixed with 0.5 mL of pepsin solution (0.2 g pepsin in 5 mL of 0.1 M HCl) and incubated on a shaking water bath for 2 h at 37uC. After 2 h of gastric digestion, the pH of digest was adjusted to 5.0. The intestinal phase of digestion was then initiated with the addition of 2.5 mL of pancreatin-bile solution (0.45 g of bile salts and 0.075 g of pancreatin in 37.5 mL of 0.1 M NaHCO3), and the digest
samples were then incubated in a shaking water bath for 2 h. To stop the intestinal digestion, the digest sample was cooled in ice for 10 min, and then the pH was adjusted to 7.2 by adding 0.5 M NaOH. The obtained digests were heated at 100uC for 4 min to inhibit the proteases. The gastrointestinal digest were centrifuged 3500gfor 1 h at 4uC. Prior to addition of rice soluble fraction to the cells, glucose (5 mM) and HEPES (50 mM) were added in order to make it similar to culture media. Deionized water was used to adjust the osmolarity to 310610 mOsm kg21 (Freezing point osmometer, Osmomat 030, Berlin, Germany). The super-natants (soluble fraction) were analyzed for Zn content and used in cell uptake assays.
Cell culture. The Caco-2 human intestinal cell line was purchased from Institute of Biochemistry and Cell Biology (SIBS, CAS, Shanghai, China) were used in experiments at passage 25– 37. The cells were grown in 75 cm2 flasks (Corning Inc., NY, USA) and maintained in high-glucose (4.5 g L21) Dulbecco’s modified minimal essential medium (GIBCO, Grand Island, NY, USA ), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY, USA), 4 mM L-glutamine,
1% (v/v) non-essential amino acids and 1% (v/v) antibiotic solution (GIBCO, Grand Island, NY, USA). The cells were incubated in a cell culture incubator (Heraeus, BB15, Germany) set at 37uC, 5% CO2 and 95% atmospheric air at constant
humidity. After reaching 80% confluence, the cells were digested by using 0.25% trypsin (GIBCO, Grand Island, NY, USA), then cells were seeded at a density of 50000 cells cm22in 1.5 mL of complete DMEM in polyester membrane chamber inserts (24 mm diameter, 0.4mm pore size; Costar Corp. NY, USA), the basal compartment contained 2.5 mL of complete Dulbecco’s modified minimal essential medium, and the medium was changed every 2 days. Zinc bioavailability experiments were carried out 21 days after initial seeding. Subsequently, the integrity of Caco-2 cells monolayer was assessed and validated by transepithelial electrical resistance (TEER) using a Millicell-ERS meter (Millipore Corpo-ration, Bedford, MA, USA). The method of TEER was according to the manufacturer’s protocols. Only those filters that had TEER values .250 V cm2 at the beginning and the end of the experiment were included. The monolayer used in this study exhibited adequate TEER values 500–680Vcm2.
Zinc uptake experiment in Caco-2 cell. Prior to the Zn bioavailability experiment, the growth medium was removed from each culture well, and the cell layer was washed three times with Ca2+and Mg2+free HBSS at 37
uC at pH 7.4. Then, 2.5 mL of transport solution (130 mM NaCl, 10 mM KCl, 1 mM MgSO4,
5 mM glucose and 50 mM HEPES, pH 7.4) was added to the bottom chamber, and 1.5 mL of rice soluble fraction was added to the apical chamber. Cell cultures were incubated at 37uC under 5% CO2, with relative humidity 95% for 2 h. After incubation,
basolateral compartrnents were collected for determine of Zn transported across the monolayer. The cell monolayer was washed twice with ice cold HBSS at pH 7.4 to remove nonspecifically bound mineral and residual medium. The cell on filters were lysed by the addition of 1 mL of deionized water in the well, and then harvested. Cell viability after 2 h of exposure to the uptake solution was assessed by trypan blue exclusion and typically 85– 95%.
The concentrations of Zn in cell retention (Zn fraction in the cell monolayer) and the solution of transport (Zn collected from basolateral compartment) were assessed by ICP-MS (Agilent 7500a, Agilent Technologies, CA, USA). Zinc uptake in Caco-2 cell model was calculated as the Zn content in cell retention plus the Zn content in transport. Solubility percentages were calculated by using following equation: solubility% = soluble fraction (mg of Zn g21sample)6100/C, where C = total Zn content of sample;
The following equation was used for Zn retention percentage: Zn retention% = Zn retention (mg well21)6100/C, where C = min-eral soluble added (mg); The following equation was used for Zn transport percentage: Zn transport% = Zn transport (mg well21)6
100/C, where C = mineral soluble added (mg); The following
equation was used for Zn uptake percentage: Zn uptake% = (retention+ transport mg well21) 6100/C, where C = mineral soluble added (mg). Due to the differences among samples in terms
of solubility of Zn afterin vitrodigestion, Zn uptake availability was expressed as Zn uptake efficiency, Zn uptake efficiency% = (% solubility6% uptake)/100. Bioavailable Zn (mg g21polished rice) = Zn concentration (mg kg21)6Zn uptake efficiency%. Quality Control of Mineral Analysis
values were in accordance with the certified ranges of 19.460.5 mg kg21 for Zn, 7.460.9 mg kg21 for Fe and 11866 mg kg21for Ca.
Statistical Analysis
Statistical analysis of the data was performed using SPSS12.0 (SPSS, Inc., Chicago, IL, USA). The data were subjected to a separate analysis of variance (ANOVA) for each cultivar, and Fisher’s least significant difference (LSD) at P,0.05 was used to determine differences between treatment means. The Pearson correlation procedure and linear regression model was used to evaluate the relationship between brown rice and polished rice Zn concentration.
Results
Biomass and Grain Yield
Biomass, grain yield, harvest index and thousand seed weight of rice did not different among the four different forms of foliar Zn treatments for all three rice cultivars (Table 2).
Zinc Concentration in Brown Rice and Polished Rice
Foliar Zn fertilization had significant (P,0.05) impact on Zn concentration in brown rice and polished rice (Fig. 1). Brown rice Zn concentration was significantly increased by foliar Zn fertilizations (Fig. 1A). Regardless of the three cultivars, Zn concentration in brown rice was increased from 30.28 mg kg21in the control, to 33.75 mg kg21by foliar Zn-EDTA application, to 35.07 mg kg21by foliar Zn-Citrate application, to 38.45 mg kg21 by foliar ZnSO4application, to 39.84 mg kg21by foliar Zn-AA
application, these represented increases of 11.46%, 15.81%, 27.26% and 31.58%, respectively. Zn concentration in polished rice was significantly increased by foliar Zn fertilizations (Fig. 1B). Regardless of the three cultivars, Zn concentration in polished rice
was increased from 22.92 mg kg21 in the control, to 25.26 mg kg21by foliar Zn-EDTA application, to 26.09 mg kg21by foliar Zn-Citrate application, to 28.08 mg kg21 by foliar ZnSO4
application, to 28.67 mg kg21by foliar Zn-AA application, these represented increases of 10.22%, 13.82%, 22.47% and 24.04%, respectively. Thus, foliar Zn fertilization could increase Zn concentration in brown rice and polished rice depending on Zn form.
The concentration of Zn in brown rice and polished rice of all cultivars were significantly increased by different forms of foliar applied Zn (Fig. 2). In control, brown rice Zn concentrations were 24.71, 30.29 and 35.82 mg kg21in cultivar Hai7, Bing91185 and Biyuzaonuo, respectively. After application of Zn-EDTA through the foliage, Zn concentrations in brown rice were 27.98, 34.47 and 38.79 mg kg21 in cultivar Hai7, Bing91185 and Biyuzaonuo, respectively. After foliar application of Zn-Citrate, Zn concentra-tions in brown rice were 28.15, 36.08 and 40.96 mg kg21 in cultivar Hai7, Bing91185 and Biyuzaonuo, respectively. After foliar application of ZnSO4, Zn concentrations in brown rice were
30.46, 38.94 and 46.20 mg kg21in cultivar Hai7, Bing91185 and Biyuzaonuo, respectively. After foliar application of Zn-AA, Zn concentrations in brown rice were 31.53, 40.62 and 47.38 mg kg21 in cultivar Hai7, Bing91185 and Biyuzaonuo, respectively (Fig. 2A). Similar trends were found in polished rice (Fig. 2B), the cultivar Biyuzaonuo had the highest Zn concentration, while Hai7 had the lowest Zn concentration in all Zn treatments.
With respect to Zn content, a significant correlation was found between polished rice and brown rice (y = 0.619x +4.232, R2= 0.897, P,0.01).
Phytic Acid Content in Polished Rice
Foliar Zn fertilization reduced the phytic acid content in the polished rice (Table 3). Regardless of cultivar, phytic acid content in polished rice ranged from 2.25 mg g21 in the control, to
Table 2.Effect of different forms of foliar Zn fertilization on the biomass, grain yield, harvest index and thousand seed weight of three rice cultivars.
Treatments Cultivarsa
Hai7 Bing91185 Biyuzaonuo Hai7 Bing91185 Biyuzaonuo
Biomass (t hm21) Grain yield (t hm21)
Control 19.50 a 18.61 a 18.35 a 7.81 a 7.98 a 7.50 a
Zn-EDTA 19.57 a 18.73 a 18.55 a 7.86 a 7.93 a 7.61 a
Zn-Citrate 19.79 a 18.66 a 18.43 a 8.00 a 8.03 a 7.53 a
ZnSO4 19.63 a 18.39 a 18.61 a 7.97 a 7.92 a 7.64 a
Zn-AA 19.14 a 18.84 a 18.80 a 8.01 a 8.04 a 7.66 a
Zn effect
by f-testb NS NS NS NS NS NS
Harvest index (%) Thousand seed weight (g)
Control 40.16 a 42.95 a 40.91 a 22.01 a 22.96 a 20.92 a
Zn-EDTA 40.20 a 42.37 a 41.05 a 23.05 a 24.13 a 21.85 a
Zn-Citrate 40.44 a 43.11 a 40.87 a 22.12 a 24.52 a 22.02 a
ZnSO4 40.59 a 43.06 a 41.13 a 22.34 a 23.68 a 22.45 a
Zn-AA 41.86 a 42.68 a 40.96 a 23.23 a 24.56 a 22.56 a
Zn effect by f-testb
NS NS NS NS NS NS
aDifferent letters after number in the same column designated significant difference by LSD P,0.05. bSignificant effects: NS = not significant at P.0.05.
2.09 mg g21 by Zn-EDTA, to 2.06 mg g21Zn-Citrate, to 1.87 mg g21by foliar ZnSO4and 1.92 mg g21by Zn-AA, these
showed that, the decreases of 6.97%, 8.42%, 16.91% and 14.57%, respectively.
Protein, Iron and Calcium Concentration in Polished Rice
Protein content in polished rice showed an increase trend by foliar Zn-AA, but not by other foliar Zn fertilizations (Table 3). Foliar application of Zn-AA had significant impact on grain protein content in cultivar Hai7 and Bing91185, except Biyuzao-nuo. Generally, compared to the control, foliar Zn-AA could increase protein content by 1.88–4.79% depending on cultivar. Grain Fe and Ca concentration did not change by different forms of Zn treatments in all three cultivars. Foliar Zn fertilization had little impact on Fe and Ca content (Table 3).
In vitroZinc Solubility of Polished Rice
The amount of Zn solubilized after in vitro digestion is an indicator for bioavailability. Foliar Zn fertilization had significant (P,0.05) effect on Zn solubility (Table 4). Averaged across the cultivars,in vitroZn solubility from polished rice was ranged from
28.48% in the control, to 29.34% by application of Zn-EDTA, 29.41% by application of Zn-Citrate, 31.15% by application of ZnSO4and 30.67% by application of Zn-AA, these represented
increase of 2.99%, 3.24%, 9.36% and 7.65%, respectively. Foliar application of Zn-AA and ZnSO4significant improvedin vitroZn
solubility in all cultivars.
Zinc Bioavailability of Polished Rice
The soluble fraction obtained fromin vitrodigestion was used to carry out the retention, transport and uptake experiments in Caco-2 cell (Table 4). Foliar Zn fertilization had significant (P,0.05) impact on the percentages of Zn retention, transport and uptake efficiency in polished rice grain for all cultivars. Generally, foliar application of Zn-EDTA, Zn-Citrate, ZnSO4 and Zn-AA could
increase the percentages of Zn retention, transport and uptake efficiency in polished rice grain, but only in case of foliar application ZnSO4 and Zn-AA could reach significant level in
most of the cultivars tested (P,0.05). Regardless of cultivar, compare to the control, after foliar application of ZnSO4, the
percentages of Zn retention, transport, and uptake efficiency from the polished rice increased by 12.96%, 31.36% and 34.98%,
Figure 1. Effect of different forms of foliar Zn fertilization on Zn concentration in rice grain.(A) Zn concentration in brown rice. (B) Zn concentration in polished rice. Error bars indicate standard errors of the means (n = 4). Different letters indicate significant difference among Zn treatments according to LSD test (P,0.05).
respectively; after foliar application of Zn-AA, the percentages of Zn retention, transport, and uptake efficiency from the polished rice increased by 19.4%, 29.47% and 35.25%, respectively. Regardless of cultivar, the amount of bioavailable Zn in the rice grain has the similar trend as the Zn uptake efficiency (Fig. 3). Regardless of cultivar, compared to the control, amount of bioavailable Zn was increased by 13.85%, 21.96%, 64.43% and 68.37% by Zn-EDTA, Zn-Citrate, ZnSO4 and Zn-AA,
respec-tively. Generally, the amount of bioavailable Zn could increased by the foliar Zn fertilizations (Zn-EDTA, Zn-Citrate, ZnSO4and
Zn-AA), but only foliar ZnSO4 and Zn-AA reach at significant
level (P,0.05) in all cultivars tested.
Discussion
The DTPA extractable Zn (Table 1) in the soil of the current field study was higher than the critical level for rice (0.8 mg kg21) [28], thus the plant was in the sufficient Zn nutritional status. In the Zn sufficient soil, excess foliar application of Zn did not affect the biomass, grain yield, harvest index and thousand seed weight (Table 2). Similar results also found in previous reports [12,29].
In contrast to grain yield, foliar Zn fertilization significantly (P,0.05) increased the Zn concentration in brown rice (Fig. 1A), consisted with the previous studies [16,30]. Thus, foliar application Zn as an effective method could boost Zn level in rice grain. Here, in this study, particular attention should be given in the Zn concentration of polished rice, as this is the predominant fraction consumed by human. In current study, we found that regardless of cultivar, although polishing process decrease substantial amount Zn from mature grain, the polished rice obtained from foliar Zn applications was still contained 10.22–24.04% more Zn than those of control (Fig. 1B), and a significant positively correlation in Zn concentration between polished rice and brown rice was also found (R2= 0.897, P,0.01), indicating that Zn concentration in polished rice might be improved by increasing the Zn concentra-tion in brown rice, consisted with previous studies [18,29], suggesting that excess foliar applied Zn could penetrate into the inner layers of rice endosperm. However, the mechanism of absorbed Zn from aleurone layer into rice endosperm still not clearly, some recent studies reported that nicotianamine and deoxymugineic acid play important role in this process [17,31].
Furthermore, the effectiveness of foliar Zn fertilization on Zn concentration of brown rice and polished rice varied with the
Figure 2. Zn concentration in rice grain among three cultivars.(A) Zn concentration in brown rice. (B) Zn concentration in polished rice. Error bars indicate standard errors of the means (n = 4). Different letters indicate significant difference among Zn treatments according to LSD test (P,0.05).
forms of Zn fertilizer (Fig. 1). Among the forms tested for foliar application, foliar Zn-AA and ZnSO4 were more effective than
Zn-EDTA and Zn-Citrate in improving the Zn concentration in brown rice and polished rice, the results agreed with the previous study [32]. The reasons might be due to the different capacity of leaf penetration of different forms of foliar applied Zn fertilizer [19]. Foliar fertilizer with low molecular weight like Zn-AA and ZnSO4might be easily penetrate into the leaves than those of
Zn-EDTA and Zn-Citrate with the high molecular weight, as a result, the more plant-available Zn from foliage might be transported and accumulated in rice grain [19,33]. The response of grain Zn concentration to foliar Zn fertilization was cultivar dependent (Fig. 2). The results consisted with the previous studies reported rice genotypes differ greatly in their response to foliar applied Zn to increase grain Zn concentration [14,16]. Thus, impact of foliar Zn application on grain Zn can be maximized by selecting genotypes with higher ability in leaf absorption and seed deposition of foliar applied Zn with low molecular weight.
Although the amount of grain Zn is important for Zn bioavailability, information about changes of anti-nutrients and other nutrients in rice grain especially polished rice during foliar Zn applications is crucial, because it related with Zn bioavailability and nutritional quality of rice, which impact on global human health. Phytic acid has long been known as a form of stored phosphorus in seeds, which was considered as an inhibitor of Zn bioavailability in rice grain [34]. In the current study, it was documented that foliar Zn applications could significantly reduced phytic acid content in polished rice, especially in case of Zn-AA and ZnSO4 applications (Table 3), consisted with the previous
studies [29,35]. Possible explanation was that foliar Zn application could inhibit the conversion of inorganic phosphorus to phytic acid in rice grain [2,35]. Iron and Ca in rice grain are also
important minerals because they are frequently deficient in human. In current study, the concentrations of Fe and Ca were varied with cultivar, but not by the foliar Zn application (Table 3). Foliar Zn fertilization had significant effect on grain protein content except the cultivar Biyuzaonuo, however, grain protein content showed increasing tread by foliar application of Zn-AA in all cultivars (Table 3), the results consisted with previous studies [12,36]. Consequently, above results indicated that foliar Zn application could reduce the phytic acid content, while could maintain the Fe, Ca and protein content in polished rice grain.
In vivosituation, Zn needs to be in soluble before it can be taken up by the enterocytes. In current study, we also determined the soluble Zn in polished rice obtained from the different foliar Zn treatments byin vitrodigestion. The results showed that regardless of cultivar, the solubility of Zn in polished rice from foliar Zn application was higher than control, especially in the case of foliar applications of Zn-AA and ZnSO4(Table 4). To the best of our
knowledge, no literature data on regarding foliar Zn fertilizers on thein vitroZn solubility in rice grain are yet available. One possible reason was thein vitroZn solubility in grain was increased by the reduction of phytic acid in grain [2,37]. The solubility method involves a simulation of gastrointestinal digestion followed by a measurement of soluble Zn in the digest and thus covers only the first phase of the overall Zn absorption process. The soluble Zn fraction obtained form in vitro digestion was used to carry out retention, transport and uptake experiment in Caco-2 cell model, which offer a more physiological tool for screening Zn bioavail-ability in food matrices [27,37,38,39]. In the current study, the percentage of Zn uptake efficiency of polished rice ranged from 6.01% to 12.33%, falling within the previous reported Zn uptake efficiency in Caoc-2 cell model from cereal foods (ranging from 4.1% to 48.1%) [37]. Compared to the control, foliar Zn-AA and
Table 3.Effect of different forms of foliar Zn fertilization on the grain protein, phytic acid, Fe and Ca contents of three rice cultivars.
Treatments Cultivarsa
Hai7 Bing91185 Biyuzaonuo Hai7 Bing91185 Biyuzaonuo
Protein (%) Phytic acid (mg g21)
Control 9.06 b 9.32 b 9.98 a 1.78 a 1.64 a 3.32 a
Zn-EDTA 9.15 b 9.34 b 9.99 a 1.66 b 1.48 b 3.14 b
Zn-Citrate 9.16 b 9.66 ab 9.91 a 1.65 b 1.47 b 3.05 b
ZnSO4 9.15 b 9.37 b 9.99 a 1.54 c 1.39 c 2.89 c
Zn-AA 9.43 a 9.76 a 10.17 a 1.50 c 1.36 c 2.67 d
Zn effect by f-testb
* * NS *** *** ***
Fe (mg kg21) Ca (mg kg21)
Control 2.92 a 3.35 a 5.89 a 92.00 a 110.22 a 146.48 a
Zn-EDTA 3.12 a 3.74 a 5.84 a 83.67 a 107.13 a 150.49 a
Zn-Citrate 3.10 a 3.81 a 5.73 a 96.38 a 112.01 a 145.90 a
ZnSO4 2.98 a 3.88 a 5.69 a 91.00 a 108.86 a 146.89 a
Zn-AA 2.98 a 3.86 a 5.79 a 94.12 a 107.43 a 143.80 a
Zn effect by f-testb
NS NS NS NS NS NS
a
Different letters after number in the same column designated significant difference by LSDP,0.05. bSignificant effects: NS = not significant at P
.0.05; *at P,0.05;
**at P,0.01; ***at P,0.001.
Table 4.Effect of different forms of foliar Zn fertilization on the percentages of solubility, retention, transported and uptake efficiency of Zn among three rice cultivars.
Treatments Cultivarsa
Hai7 Bing91185 Biyuzaonuo Hai7 Bing91185 Biyuzaonuo
Solubility (%) Retention (%)
Control 29.17 c 30.58 c 25.70 b 14.53 c 14.91 b 14.39 c
Zn-EDTA 30.75 c 31.21 bc 26.06 b 14.82 bc 14.96 b 15.04 bc
Zn-Citrate 30.90 bc 31.13 bc 26.20 b 14.93 bc 15.78 b 15.11 bc
ZnSO4 32.68 a 32.24 a 28.54 a 16.68 ab 18.29 a 16.80 ab
Zn-AA 31.64 ab 32.62 a 27.73 a 17.54 a 18.80 a 17.53 a
Zn effect by f-testb
* * *** * *** *
Transport (%) Uptake efficiency (%)
Control 9.35 b 16.09 b 8.99 c 6.95 c 9.48 b 6.01 b
Zn-EDTA 9.61 b 16.23 b 9.57 c 7.52 bc 9.73 b 6.39 b
Zn-Citrate 11.08 ab 16.15 b 9.96 bc 8.02 b 9.94 b 6.57 b
ZnSO4 13.27 a 18.43 ab 13.53 a 9.79 a 11.84 a 8.66 a
Zn-AA 13.05 a 19.11 a 12.42 ab 9.68 a 12.39 a 8.30 a
Zn effect by f-testb
** * * *** *** ***
a
Different letters after number in the same column designated significant difference by LSDP,0.05. bSignificant effects: NS = not significant at P
.0.05; *at P,0.05;
**at P,0.01; ***at P,0.001.
doi:10.1371/journal.pone.0045428.t004
Figure 3. The amount of bioavailable Zn in the polished rice among three cultivars.Error bars indicate standard errors of the means (n = 4). Different letters indicate significant difference among Zn treatments according to LSD test (P,0.05).
ZnSO4 could significantly increase the percentages of Zn
retention, transport and uptake efficiency of polished rice in most of the cultivars tested (Table 4). Regardless of cultivar, the amount of bioavailable Zn has the similar trend as Zn uptake efficiency (Fig. 3), showing the foliar Zn could improve the amount of bioavailable Zn in the polished rice, especially foliar Zn-AA and ZnSO4could increased by 68.37% and 64.43%, respectively. The
results indicated that foliar Zn application could improve the bioavailability of Zn from polished rice but depended on the forms of Zn application. Till now, few literatures on the Zn bioavail-ability of biofortified polished rice obtained from the foliar Zn fertilization are available. The possible explanation of our results might be foliar Zn-AA and ZnSO4have the higher efficiency to
decrease the phytic acid, and improve the total amount of Zn than Zn-EDTA and Zn-Citrate, as a result, increase the amount of bioavailable Zn in the polished rice grain. In addition, in the current study, we also observed that the polished rice of cultivar Biyuzaonuo contained the highest amount of Zn, interestingly, it was not contained the highest amount of bioavailable Zn, this is might be due to presence of significant amount of phytic acid or other anti-nutrients in this cultivar [40]. Thus, it is suggested that not only the net grain Zn concentration but also the bioavailability of grain Zn should be considered in ongoing breeding program.
In conclusion, foliar Zn fertilization was an effective agronomic practice to promote grain Zn concentration and Zn bioavailability, especially, in case of Zn-AA and ZnSO4.On average, Zn-AA and
ZnSO4increased Zn concentration in polished rice up to 24.04%
and 22.47%, respectively. On average, Zn-AA and ZnSO4
increased Zn bioavailability in polished rice up to 68.37% and 64.43%, respectively. The effectiveness of foliar applied Zn-AA and ZnSO4 were higher than Zn-EDTA and Zn-Citrate to
improve the Zn concentration, and reduction of phytic acid, as a results higher accumulation of bioavailable Zn in polished rice. Moreover, foliar Zn application could maintain the protein and minerals (Fe and Ca) quality of the polished rice. Therefore, it’s believed that foliar application of suitable Zn form is a feasible approach to improve the bioavailable Zn status in polished rice.
Acknowledgments
The authors gratefully thank Dr. Zed Rengel (The University of Western Australia) for critical comments on the manuscript.
Author Contributions
Conceived and designed the experiments: YW MJIS XY. Performed the experiments: YW MJIS. Analyzed the data: YW MJIS XY. Contributed reagents/materials/analysis tools: YW MJIS. Wrote the paper: YW MJIS.
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